Diffractive Sensor for Sensing Target Analytes in a Sample, and System and Method for Sensing Target Analytes in a Sample by Said Diffractive Sensor

The present invention relates to a sensor that exploits the optical phenomenon of diffraction by diffractive gratings for the detection of target analytes in a sample. The present invention also relates to a method and a system for sensing target analytes in a sample that exploit such a diffractive sensor.

The term “target analyte” refers to any chemical species whose presence in a sample is to be determined.

The present invention has particular application for the detection of target analytes, such as viruses or bacteria or their components, but can also be applied for the detection of target analytes of other kinds, thus not only in the medical, veterinary, and diagnostic fields, but also for example in the biosafety or chemical fields, especially for the detection of traces of contaminants.

The detection of target analytes can be carried out according to numerous criteria and technologies, which depend on the nature of the analyte itself.

For example, reliable diagnosis of infections, performed through the detection of viral or bacterial infectious agents, is generally carried out by means of more or less complex tests, which usually require the use of sophisticated laboratory equipment and involve long times, even of the order of a few days, before results are available. Alternatively, rapid tests are also available, but these have considerably lower sensitivity than the above-mentioned tests, and thus can lead to outcomes that are not entirely reliable and are problematic from a clinical point of view.

There is therefore a particular need to make available devices that enable the detection of viruses or bacteria, as well as other target analytes, in a simple, fast, reliable, and reproducible manner.

For example, EP 3907507 A1 describes a colorimetric sensor comprising a functional layer with a nanomaterial capable of generating a surface plasmon that is bioresponsive to bacteria and/or viruses, a receptor layer comprising protein substances or antibodies functioning as virus receptors, and a plasmonic nanostructured layer comprising etched nanostructures such that plasmonic colours are generated. When secretions from an individual containing the virus or bacterium are placed in contact with the functional sensor layer, the change in the latter's structure causes the change in plasmon energies and thus in the colours perceived. The change in coloration is thus indicative of the presence of the virus or bacterium in the secretion.

The object of the present invention is to provide a sensor alternative to those according to the known art that enables with simplicity, rapidity, and reliability the detection of generic target analytes, such as bacteria or viruses.

1 17 22 This and other objects are achieved by a diffractive sensor for sensing a target analyte according to claim, a system for sensing a target analyte according to claim, and a method for sensing a target analyte according to claim.

Dependent claims define possible advantageous embodiments of the invention.

1 4 FIGS.- 1 1 With reference to the attached, a diffractive sensor for sensing a generic target analyte (or, as will be seen, a plurality of target analytes) is referred to as a whole as. Sensorcan, for example, be made in the form of a label to be affixed to a user instrument (not shown in the figures).

1 2 2 Sensorpreferably comprises a support layer, having the function of supporting additional overlying layers, as will be described in detail below. The support layeris preferably transparent or semi-transparent and can be made of, but not limited to, polycarbonate, or PVC, or Teslin, or polyester, or the like.

1 3 2 Sensorcomprises a diffractive layer, preferably applied, either directly or indirectly, on the support layer.

3 30 Diffractive layercomprises a diffractive gratingnanostructured, that is, provided with diffractive structures having a depth of the order of a few tens to a few hundred nanometers, as will be described in detail below.

30 on a polymer film, such as a polyester film (such as polycarbonate PC, polyethylene terephthalate PET, polyvinyl chloride PVC, polypropylene PP), preferably having a thickness comprised between 5 μm and 500 μm; in an amorphous (e.g., a glass) or highly crystalline (e.g., a quartz) oxide material, with a thickness preferably comprised between 10 μm and 500 μm; in a fiberglass material. Diffractive gratingcan be realized:

30 ablation: this technique involves the selective removal of material and includes, for example, pulsed laser techniques such as the LIPSS (Laser-induced periodic surface structures) technique, or the LEHCEB (Low Energy High Current Electron Beam) technique; deposition: this technique involves the deposition of thin material on a surface and includes, for example, sputtering and optical photolithography. For example, diffractive gratingcan be made by one of the following techniques:

3 40 30 40 Diffractive layercomprises a plurality of surface regionsthat are equal to each other, that is, in which the diffractive gratinghas the same conformation. Furthermore, surface regionshave same shape and dimensions.

40 40 Surface regionscan have any shape, and, according to a possible embodiment, they have a square contour. Surface regionspreferably have a maximum dimension (identifiable as the maximum distance between two points of the contour, coinciding with a single side of the square in the case of the square contour cell) comprised between 5 μm and 50 μm, still more preferably between 30 μm and 45 μm, e.g., equal to 40 μm.

40 2 FIG. 3 FIG. Surface regionsmay be arranged side by side (for example, in the case of square surface regions, they may have a checkerboard arrangement, as illustrated in), and/or they may be partially overlapping each other (in other words, a vertex of one surface region may fall within another surface region, as illustrated in).

40 30 40 30 30 4 FIG. 4 FIG. Referring now to a single surface region(), diffractive gratinghas grooves that form a pattern having preferentially a random pattern, which, however, is repeated equally in each surface region. As visible in, in which the darker parts represent the valleys and the lighter parts the peaks of the grooves of diffractive grating, no geometrically defined pattern formed by the grooves can be identified (in this sense, the pattern of the grooves of diffractive gratinghas a “random” pattern).

30 1 4 30 4 sulfides, such as zinc sulfide (ZnS) 2 2 2 oxides, such as titanium oxide (TiO), zinc oxide (ZnO), zirconium oxide (ZrO), and silicon oxide (SiO); metals, such as gold (Au), silver (Ag), nickel (Ni), zinc (Zn), aluminum (Al), and cop-per (Cu). The groove depth of diffractive grating(i.e., the distance between the peaks and valleys) is less than 200 nm, preferably comprised between 30 nm and 200 nm, still more preferably between 100 nm and 180 nm. In an embodiment, diffractive sensorfurther comprises a protective layerto protect diffractive grating, preferably having a thickness comprised between 1 μm and 100 μm. The protective layercan be made of a material selected from the group consisting of:

4 30 The protective layeris preferably made by deposition (e.g., under vacuum) of nanoparticles of the above-mentioned materials on the diffractive grating. Preferably, the nanoparticles are comprised between 4 and 30 nm in size, still more preferably smaller than or equal to 20 nm, e.g., equal to 8 nm.

30 3 100 1 2 1 200 5 FIG. The diffractive gratingof the diffractive layerconformed as mentioned above is such that, if a beam of monochromatic, polarized light (LASER) emitted by a laser sourceplaced posterior to the sensoritself (i.e., on the side of the sensor where the support layeris located) and preferably incident at 90° is passed through the sensor, this beam of light is diffracted into a diffraction image visible to the naked eye on a screen, without the need to interpose (between laser and sensor, as well as between sensor and screen) optical means, such as filters, or lenses, to amplify the image (), according to the well-known Bragg's law:

n is a positive or negative integer (diffraction order); λ is the wavelength of the LASER beam; 1 200 d is the distance between sensorand screenonto which the diffracted image is projected θ is the diffraction angle of order 1, which depends on the conformation of the diffractive grating. wherein: nλ=2dsinθ

In other words, due to the conformation of the nanometric diffractive grating, a diffractive phenomenon takes place at the macroscopic level, i.e., visible to the naked eye, similar to that which takes place, for example, at the atomic level following exposure of matter to X-rays, a phenomenon which, however, is not detectable to the naked eye.

100 1 As an example, the laser light emitted by laser sourcecan have wavelength A equal to 532 nm (green light), however the diffraction image can be generated by irradiating sensorwith a laser light having any other wavelength in the visible spectrum (indicatively comprised between 390 nm and 700 nm).

200 30 40 6 FIG. An example of a diffraction image that is produced on screenis shown in. It comprises a plurality of dots whose distribution depends on the conformation of the diffractive gratingrepeated in the surface regions. In the figure, a larger dot can be seen, which is the dot aligned with the laser light incident at 90° posterior to the sensor, which passes through it without being diffracted.

40 30 1 It is to be observed that, given the periodicity of the surface regions, which are repeated equally in the diffractive layer, the diffraction image is generated no matter where on the sensorthe laser light beam is aimed.

4 200 4 40 1 1 30 The conformation of the protective layercan affect the brightness of the dots in the diffraction image on the screen. For example, it has been observed that employing zinc nanoparticles in the protective layerresults in a brighter diffraction image than if gold nano-particles are used, but the distribution of dots in the diffraction image remains the same. There-fore, the conformation of the diffractive gratings in the surface regionscauses sensorto produce a kind of unique fingerprint of sensor, that is, precisely the diffraction image. Changing the random pattern conformation of the diffractive gratingwill also result in a different distribution of dots in the diffraction image.

3 It is to be observed that diffractive layeris conformed so that the light diffracted by the sensor is polarized.

4 It should also be noted that the diffraction image can be obtained not only by crossing the sensor by the laser beam from the back side, as described above, but also by reflection of the same, i.e., by pointing a laser beam at an angle to the front surface of the sensor (i.e., on the side opposite to the bottom side, for example, on the side where the protective layermay be arranged). In both cases, a diffraction image having the characteristics said above is obtained.

1 FIG. 1 5 3 4 Returning now to, diffractive sensorfurther comprises a receptor layerover-lapping and directly in contact with diffractive layer, or overlapping and directly in contact with protective layer, where present.

5 5 Receptor layeris able to bind selectively to the target analyte to be detected and not to substances of a different nature. The target analyte can be contained in a sample, such as a biological solution (e.g., a saliva or blood or urine sample), which can be deposited, for example smeared or rubbed, on receptor layer.

5 1 5 1 5 5 1 7 FIG. 7 a FIG. 7 b FIG. In this way, if the target analyte is not present in the sample, receptor layeris not altered and diffractive sensor, when subjected to a laser light beam as described above, produces a first diffraction image on the screen, visible to the naked eye. On the other hand, when the target analyte is present in the sample, it binds to receptor layer, and as a result diffractive sensorproduces a second diffraction image different from the first, again visible to the naked eye, and comparable with the diffraction image of the first condition.shows a comparison between a diffraction image produced in the case of absent target analyte ()) and in the case of target analyte present and bound to receptor layer()). As can be seen, the number and/or distribution and/or light intensity of the dots visible in the diffraction image of the first and second cases are different. Therefore, from the simple visual comparison, which can also be made with the naked eye, of the two diffraction images, it is possible to determine whether the target analyte is present in the sample and has bound to receptor layerof diffractive sensor, or not.

3 5 5 3 4 5 In general, the diffraction image produced by diffractive layeralone is different from the diffraction image produced as a result of the application of receptor layerand is also different from the diffraction image produced in the case where receptor layerbinds to the target analyte. Therefore, the sensor comprising diffractive layer, with or without protective layer, and lacking receptor layer, is as such capable of producing a diffraction image which is peculiar to the nanometer diffractive grating, which constitutes a kind of fingerprint of the sensor.

It should be observed that the diffraction images produced in the presence and absence of the target analyte may also differ from each other in coloration. However, this is a possible secondary effect to the variation in the diffraction image. Preferably, the diffraction image, whose colour depends on the laser light used (e.g., for a wavelength λ=532 nm, the colour is green), is converted to a gray scale.

5 5 It should also be noted that, in order to prevent any pollutants other than the target analyte, which are present in the sample and/or in the environment where the sensor is located, from altering the diffraction image, it is preferable that receptor layer, following the application of the sample where the possible target analyte presence is to be detected, is washed in an appropriate manner, some possible examples of which will be given below. In this manner, substances or molecules or contaminants, which cannot bind to receptor layer(which is able to selectively bind only to the target analyte), are removed.

5 5 The nature of receptor layer, in particular its chemical composition, as well as the manner of washing (where applicable), may differ depending on the target analyte being sought. In general, receptor layermay include antibodies, proteins, molecules with key-lock action, chelating compounds or other chemical or biological substances that have chelating functions.

5 1 human chorionic gonadotropin (hCG): in this case receptor layermay include a monoclonal antibody specific for hCG, and sensorwill be suitable for pregnancy testing; 5 1 Tetanus toxoid: in this case receptor layermay include a monoclonal antibody specific for tetanus toxin, and sensorwill be able to detect tetanus toxin; 5 1 nucleocapsid (N) protein of SARS-CoV-2: in this case, receptor layermay include a monoclonal antibody specific to the nucleocapsid protein of SARS-CoV-2, and sensorwill be able to detect the presence of SARS-CoV-2 virus in a biological sample. By way of non-limiting example, target analytes may include:

5 5 Therefore, the inclusion in receptor layerof the above-mentioned antibodies enables the latter to selectively detect the presence of these target analytes in the sample that is applied onto receptor layer. In general, a suitable antibody against a component to be detected by the sensor can also be specifically produced.

1 3 4 In general, in the case where the target analyte is a specific antigen, the receptor layer may include the antibody specific for that antigen and nonspecific for antigens other than the target antigen, which is intended to be detected by the sensor. It is therefore necessary for the anti-body to be firmly bound to the diffractive layerfor the detection to be reliable. Advantageously, such firm binding can be achieved by binding the antibody to the nanoparticles, preferably metallic, of the protective layer.

Antibodies are modular-structured protein complexes with a common basic structure but showing variability in specific regions capable of binding to specific antigens. In general, anti-bodies exhibit a Y conformation, with a central stem and two side branches. They comprise four chains covalently linked by —S—S — disulfide bridges and are unable to bind to a metal, such as gold, unless processed in advance.

4 4 10 8 a FIGS. 8 b FIG. 9 FIG. d One possible pretreatment of the antibody for the purpose of its binding to the protective layer, in particular to the metal nanoparticles thereof, involves breaking the —S—S— disulfide bond of a portion of the antibody by reducing it to a reduced —SH disulfide bond, which is instead capable of binding to the metal, in particular forming very strong thiol bonds with the latter. If the —S—S— disulfide bonds of the antibody are broken, four different portions of the antibody are obtained ()-)), in which only half of the whole antibody (shown in)), i.e., the portion of the antibody that includes a single side branch i.e., a Fab′ fragment, and half of the stem, i.e., half of the Fc fragment, is suitable to form a stable bond with the metal of the protective layeron the one hand and to maintain the ability to bind to the target antigenon the other (). Disulfide —S—S— bonds are then reduced in a hinge region that holds together the two heavy chains of the antibody, which are separated.

Since all antibody portions obtainable by breaking disulfide —S—S— bonds differ in size, it is possible to select the desired antibody portion, for example, by filtering precisely according to size.

Rafael Antonio Salinas Domínguez, Miguel Ángel Domínguez Jiménez, Abdú Orduña Díaz: “Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection”—ACS Omega 2020, 5, 20473-20480; Anke K. Trilling, Jules Beekwildera, Han Zuilhof: “Antibody orientation on biosensor surfaces: a minireview”—Analyst, 2013, 138, 1619; Lu Zhang, Yacine Mazouzi, Michèle Salmain, Bo Liedberg, Souhir Boujday: “Anti-body-Gold Nanoparticle Bioconjugates for Biosensors: Synthesis, Characterization and Selected Applications”—Biosensors and Bioelectronics 165 (2020) 112370; Asta Makaraviciute, Carolyn D. Jackson, Paul A.Millner, Almira Ramanaviciene: “Considerations in producing preferentially reduced half-antibody fragments” Journal of Immunological Methods 429 (2016) 50-56; Monalisa Pal, Sanghee Lee, Donghoon Kwon, Jeongin Hwang, Hyeonjeong Lee, Seokyung Hwang, Sangmin Jeon: “Direct immobilization of antibodies on Zn-doped Fe3O4 nanoclusters for detection of pathogenic bacteria”—Analytica Chimica Acta 952 (2017) 81-87; Md. Morsaline Billah, Christopher S. Hodges, Henry C. W. Hays, P. A. Millner: “Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.”—Bioelectrochemistry 80 (2010) 49-54; Hongcheng Liu & Kimberly May (2012): ‘Disulfide bond structures of IgG molecules’—mAbs, 4:1, 17-23; Roberto Reverberi, Lorenzo Reverberi: “Factors affecting the antigen-antibody reaction”—Blood Transfus 2007; 5:227-240; Harsh Sharma, Raj Mutharasan: “Half Antibody Fragments Improve Biosensor Sensitivity without Loss of Selectivity”—Anal. Chem. 2013, 85, 2472-2477; Woochang Lee, Byung-Keun Oh, Won Hong Lee, Jeong-Woo Choi: “Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance.”—Colloids and Surfaces B: Biointerfaces 40 (2005) 143-148; Yuanli Song, Hui Cai, Zhijun Tan, Nesredin Mussa, Zheng-Jian Li: “Mechanistic insights into inter-chain disulfide bond reduction of IgG1 and IgG4 antibodies”—Applied Microbiology and Biotechnology (2022) 106:1057-1066; Nicholas G. Welch, Judith A. Scoble, Benjamin W. Muir, et al: “Orientation and characterization of immobilized antibodies for improved immunoassays”—Biointerphases 12, 02D301 (2017); Arkady A. Karyakin, Galina V. Presnova, Maya Yu. Rubtsova, Alexey M. Egorov: “Oriented Immobilization of Antibodies onto the Gold Surfaces via Their Native Thiol Groups”—Anal. Chem. 2000, 72, 3805-3811; Nur Mustafaoglu, Tanyel Kiziltepe, Basar Bilgicer: “Site-Specific Conjugation of Antibody on Gold Nanoparticle Surface for One-Step Diagnosis of Prostate Specific Antigen with Dynamic Light Scattering”—Nanoscale. 2017 Jun. 29; 9(25): 8684-8694; Harry W Schroeder Jr, Lisa Cavacini: “Structure and Function of Immunoglobulins”—Allergy Clin Immunol. 2010 February; 125(2 0 2): S41-S52. The reduction of —S—S— disulfide bonds to reduced —SH disulfide bonds can be accomplished by molecular techniques known per se (such as in particular: use of reducing agents, polyacrylamide gels, ultrafiltration devices). In this regard, see, for example, the following papers, the contents of which are fully incorporated herein by reference:

5 In cases where receptor layercomprises antibodies or other proteins, the previously mentioned washing can, for example, be done by the use of PBS (Phosphate Buffered Saline), by immersion and possibly by subsequent centrifugation of the sensor.

5 5 5 5 It should be noted that, according to one possible embodiment, receptor layeris able to selectively bind to a plurality of different target analytes, for example in different areas of receptor layer, and is configured such that that, depending on the bound target analyte, the diffraction image produced is different. For example, receptor layermay include different antibodies positioned in such a way that receptor layeris able to selectively bind to different target antigens

5 FIG. 100 200 1 300 200 400 Although the diffraction images produced by the sensor are visible to the naked eye, they can be usefully detected by an automated or partially automated system. As an example, a possible system for sensing a target analyte in a sample is shown in, which comprises laser source, screen(preferably satin), and a vision system capable of acquiring the diffraction image produced by sensor, e.g., a video camerapointed at screen, connected to a control unit of, for example, a computer. The latter control unit can possibly perform gray-scale conversion of the diffraction image. Such a system may be portable, e.g., to perform on-site analysis. In such a case, power supplies and/or batteries may be provided for the above-mentioned equipment.

1 providing a diffractive sensorin accordance with the embodiments described above; 5 1 applying the sample to the receptor layerof the diffractive sensor; 1 100 1 200 hitting the diffractive sensorwith a beam of laser light-for example, emitted by source-so that diffractive sensorproduces a diffraction image visible to the naked eye-for example, on screen; 1 5 400 comparing the diffraction image produced by the diffractive sensorwith a reference diffraction image obtained by hitting the diffractive sensor with an equal beam of laser light in the absence of the target analyte on the receptor layer. This comparison can be made by a naked-eye operator or, preferably, by an automated control unit, e.g., of the computer; 1 determining the presence of the target analyte in the sample if the diffraction image produced by diffractive sensoris different from the reference diffraction image. According to a further aspect of the present invention, a method for sensing a target analyte in a sample comprises the steps of:

In an embodiment, the method comprises a step of converting diffraction images to a grey scale, to remove the coloration that depends on the wavelength of the laser light beam.

This step can be carried out, for example, by the control unit.

In the case where the receptor layer is able to selectively bind to a plurality of target analytes, the method may further comprise the additional steps of comparing the diffraction image produced by the sensor with a plurality of stored diffraction images (each corresponding to a specific target analyte), and determining the presence of a specific target analyte from the plurality of target analytes detectable by the sensor if the diffraction image produced by the sensor matches the stored diffraction image for that specific target analyte.

5 1 1 5 In an embodiment, following the step of applying the sample to receptor layerand prior to the step of hitting diffractive sensorwith a laser light beam, the method comprises an additional step of washing diffractive sensorto remove substances or molecules or contaminants other than the target analyte from receptor layerthat might interfere with the final diffraction image, even if only as background noise. Such a washing step can, for example, be carried out by using buffer solutions (e.g., PBS), ionic or non-ionic detergents, or by mild surfactants, according to appropriate procedures and timing.

diagnosis, by demonstration of the pathogen or its components, of bacterial (such as: Lyme disease, Brucellosis, Syphilis), viral (such as: HIV, Hepatitis A,B,C) and fungal (such as pathogenic yeast infections) infections; detection of viruses (such as: Coronavirus, HIV, Hepatitis, Ebola, Norovirus, Influenza, West Nile, Zika); diagnosis, by demonstration of specific clinically validated markers, of auto-immune diseases, due to abnormal production by the immune system of autoantibodies that destroy cells in the body (for example: autoantibodies that destroy insulin-producing cells in the pancreas in type I diabetes); detection and quantification of hormones (e.g., human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), testosterone); screening of donated blood (e.g., for detection of viral infections, such as HIV); detection of drugs (for example: amphetamines, cocaine); detection of tumoral markers (for example: prostate specific antigen (PSA) for prostate cancer diagnosis); diagnostics in the veterinary field, for example: detection of African swine fever, avian fever, bovine parvovirus, canine adenovirus, coronavirus, equine infectious anemia, feline leukemia, etc.). Diagnostics in medical or veterinary settings: DNA search; Surveillance of epidemics; Biological weapons/bioterrorism testing; Testing in cosmetics (for example: testing for metal contamination); Biosafety: Salmonella Escherichia coli, Campylobacter Staphylococcus aureus Detection and screening of contaminants in food (eg:sp.,sp.,); Detection and screening of environmental pollutants (e.g., detection of heavy metals in water or soil); Pesticide detection; Gunpowder detection (for example: nitrate detection by specific en-zymes, such as nitrate reductase). Detection of contaminants: Non-diagnostic applications: The diffractive sensor, system and method according to the present invention can find application in a variety of fields, of which some non-limiting examples are given below:

1 It should be noted that in this description and the attached claims the term “overlapping,” referring to the layers of sensor, is not intended to imply necessarily also a direct contact between the mentioned overlapping layers. Such layers may therefore be in direct contact with each other, or, alternatively, they may have one or more intermediate layers arranged between them, without prejudice to their overlapping.

It should also be noted that, for the purpose of this description and the appended claims, except where otherwise indicated, all numbers expressing quantities, measures, percentages, and so on, are to be considered as modified in all cases by the term “about.” Further, all ranges include any combination of the maximum and minimum points disclosed and include any inter-mediate ranges within them, which may or may not be specifically enumerated.

This disclosure, in accordance with at least one of the above aspects, may be implemented in accordance with one or more of the described embodiments, optionally combined with each other.

For the purpose of this description and the attached claims, the terms “an” or “one” should be read as including one or at least one and the singular includes the plural, unless it is obvious that it is intended otherwise.

To the description given above of the diffractive sensor and the system and method for sensing a target analyte in a sample the skilled person, in order to meet specific contingent needs, may make numerous additions, modifications, or substitutions of elements with functionally equivalent ones, without, however, departing from the scope of the attached claims.